Size-Exclusion Chromatography

Size-exclusion chromatography (SEC), also known as gel filtration chromatography, is a widely used analytical technique in the field of biochemistry, biotechnology, and pharmaceutical sciences. It is a liquid chromatography method that separates molecules based on their size and shape, making it particularly useful for separating macromolecules such as proteins, nucleic acids, and polysaccharides.

The principle of size-exclusion chromatography relies on the differential partitioning of molecules between a stationary phase, typically a porous gel matrix, and a mobile phase, which is usually a buffered solution. The stationary phase consists of spherical beads with controlled pore sizes. These beads can be made from various materials such as agarose, dextran, or polyacrylamide, and they are often crosslinked to enhance their stability and durability.

The separation process begins with the sample mixture being injected onto the top of the column, which is packed with the stationary phase beads. As the mobile phase (buffer) is continuously pumped through the column, it flows around and through the porous beads. The separation is achieved based on the size of the molecules, as smaller molecules can penetrate the pores and thus spend more time inside the beads, resulting in slower elution from the column. On the other hand, larger molecules are excluded from entering the pores and, therefore, experience a faster flow through the column.

The separation mechanism in SEC is often referred to as “sieving.” The porous nature of the stationary phase allows smaller molecules to enter the beads and diffuse into the interior, resulting in increased residence time within the column. In contrast, larger molecules are unable to enter the pores due to their size, and they instead flow around the beads, taking a shorter path and eluting more quickly. This differential behavior of molecules based on their size leads to the separation of the sample components.

The efficiency of the separation depends on the choice of the stationary phase and its pore size distribution. Columns with larger pore sizes are suitable for separating larger molecules, while those with smaller pore sizes are used for smaller molecules. The pore size of the stationary phase is typically characterized by a parameter called the “exclusion limit,” which represents the size of the largest molecule that can enter the pores and be retained within the matrix.

Detection of the separated molecules in SEC can be achieved using various methods, including UV-visible spectroscopy, refractive index measurement, fluorescence detection, or light scattering. UV-visible spectroscopy is commonly employed for monitoring proteins and nucleic acids, as they often exhibit absorbance at specific wavelengths. Refractive index detection is useful for detecting non-absorbing molecules, while fluorescence detection is employed when the target molecules have fluorescent properties. Light scattering detection is particularly valuable for determining the absolute molecular weight of the separated molecules.

SEC is a versatile technique that offers several advantages. It is a non-destructive method that can be used under mild conditions, preserving the integrity and activity of the analyzed molecules. It can separate molecules ranging from small ions to large polymers, making it suitable for a wide range of applications. SEC can also be used for desalting or buffer exchange of samples, as smaller molecules such as salts or low-molecular-weight contaminants are efficiently washed out of the column while the target molecules are retained.

So there we have it, size-exclusion chromatography is a powerful analytical technique for separating molecules based on their size and shape. By exploiting the differential partitioning of molecules between a stationary phase with controlled pore sizes and a mobile phase, SEC enables the separation and characterization of macromolecules in complex mixtures. Its broad applicability, non-destructive nature, and compatibility with various detection methods make it an indispensable tool in biochemistry, biotechnology, and pharmaceutical sciences.

Article amended from 2nd January 2004 with new information on the technology.